The prominent role of aberrant transcriptional regulation in oncogenesis is underscored by the frequent occurrence of non-random, disease specific mutations in genes encoding transcription factors. One such mutation, the t(17;19) observed in ALL, creates an E2A-HLF fusion protein with properties of a chimeric transcription factor. Functionally analogous chimeras are frequently created by other translocations in acute leukemias and childhood sarcomas. Understanding the mechanisms by which aberrant transcriptional regulation contributes to malignant transformation is an essential prerequisite to developing new therapies directly targeted at the molecular events mediating oncogenesis. The proposed studies will be performed in parallel in two experimental system in which E2A-HLF has biological activity relevant to its role in ALL: blocking apoptosis induced by growth factor withdrawal in the murine IL3-dependent pro-B cell line FL5.12 and transformation of NIH 3T3 cells. Specific Aim 1 is designed to rigorously examine the hypothesis that transcriptional activation of crucial target genes is the major mechanism for E2A-HLF biological activity. Point mutations that inactivate well-defined domains involved in transcriptional regulation will be used to delineate E2A-HLF functions that are necessary for biological activity. Functional properties which are sufficient for biological activity will then be defined by replacing necessary domains with heterologous domains capable of accomplishing the same function. The second aim of this proposal is to identify target genes transcriptionally regulated by E2A-HLF and evaluate their role in leukemogenesis. The ligand binding domain of the estrogen receptor (ER) has been fused to E2A-HLF to create a conditionally active transcriptional activator, E2A-HLF-ER. 3T3 and FL5.12 cells will be stably transfected with E2A-HLF-ER and a transcriptionally inactive mutant construct and representational difference analysis will be used to identify mRNAs differentially expressed following induction of E2A-HLF-ER activity. Candidate mRNAs which are also expressed in t(17;19)+ human leukemias will be further characterized to define the role of their protein products in leukemogenesis.